Giardia is a widespread zoonotic intestinal parasite that causes acute and chronic diarrheal disease in more than 280 million people each year. Motile trophozoites colonize and attach to the small intestine with the ventral disc, a complex microtubule organelle. Attachment is required for infection as it allows Giardia to resist peristalsis. Theoretical models of attachment must be grounded in accurate biological data. For 50 years, hydrodynamic suction has been extensively modeled as the leading mechanism for Giardia attachment, yet this model of attachment lacks empirical support. Our pioneering work on disc architecture and composition, combined with our development of CRISPR-mediated knockdowns and knockouts and bioluminescent imaging of infection dynamics in animals, enable us to genetically test the structural and/or functional roles of DAPs required for disc conformational dynamics in attachment. During early stages of attachment, we discovered that regions of the disc undergo specific conformational changes. These changes, along with the presence of a curved disc, likely create a ?seal? that enables attachment to the surface and resistance to shear forces. We have also recently identified and localized 87 disc-associated proteins (DAPs) to the specific structural and functional regions of the disc involved in maintaining and modulating disc conformations. Here we use our new CRISPR-based genetic tools to create specific classes of DAP mutants associated with key regions of the ventral disc that are likely required for its domed structure, as well as DAP mutants in key regions associated with flexible movements. We evaluate structural and attachment defects in CRISPR-interference (CRISPRi) DAP knockdowns (10 total) using high-resolution live imaging, electron microscopy, and biophysical assays. In Aim 1, we interrogate the role of the conspicuous microribbon and crossbridge complexes of the disc in mediating disc curvature. In Aim 2, we define the structural and functional roles of overlap zone DAPs that may structurally link the upper and lower portions of the disc, enabling proper domed conformation. In Aim 3, we interrogate the molecular mechanisms of DAPs associated with disc margin and ventral groove movements that contribute to formation of the lateral crest seal needed to resist shear forces. Lastly, we evaluate aberrant infection dynamics of two disc structural mutants using in vivo bioluminescent imaging (BLI) in an animal model of infection. Therapies that target parasite attachment would limit host colonization and limit the dissemination of infectious cysts.